Acrosin (ACR), a serine proteinase located in the acrosome of the sperm, has been presumed to be involved in the recognition and binding of the sperm to the zona pellucida of the ovum and the sperm penetration through the zona pellucida. To examine the function of acrosin in vivo, we have generated mice carrying a mutation at the acrosin locus (Acr) through targeted disruption in embryonic stem (ES) cells. One chimeric male and female transmitted the targeted gene through their germ line. Homozygous Acr-/- mice are fertile and yield litters comparable in number and size to those of Acr+/+ mice. These data show that sperm of the homozygous Acr-/- mice are able to penetrate the zona pellucida, fertilize the ovum, and produce viable offspring. However, spermatozoa lacking acrosin protein show a delayed fertilization. One chimeric male which contained the targeted gene in 20% of its sperm transmitted only the Acr+ allele to its progeny. Furthermore, in vitro fertilization with equally mixed sperm cells of Acr+/+ and Acr-/- mice resulted in fertilization only with the Acr+ sperm cells. Incubation of oocytes with Acr+ or Acr- sperm show that the Acr+ sperm are faster to fertilize the oocytes than the Acr- sperm cells. These results suggest that Acr- sperm have a selective disadvantage when they are in competition with Acr+ sperm.